Details of a Researcher - Tomita, Masaru

Papers - Tomita, Masaru

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Phylogenetic position of a rare loricated green alga, Cephalomonas granulata N. L. Higinb. (Volvocales, Chlorophyceae)

Nakada, T., Soga, T. and Tomita, M.

Phycol. Res. 58 ( 1 ) 62-68 - 68 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 13220829

Cephalomonas (Cp.) granulata N. L. Higinb. (Volvocales, Chlorophyceae), a rare volvocalean phytoflagellate, has recently been isolated from a paddy field in Japan and examined by light and fluorescence microscopy. The vegetative cells of Cp. granulata have granulate loricae and a characteristic mushroom-like shape with a hemispherical to spherical anterior part and narrower posterior part. Cp. granulata has been classified among the Phacotaceae along with other loricated genera. However, its phylogenetic position has not been investigated using molecular phylogeny. To evaluate the phylogenetic position of Cephalomonas, the 18S rRNA gene sequence of the Japanese strain was determined. The phylogenetic analyses revealed that Cp. granulata was not closely related to other loricated taxa with known phylogenetic affinities, such as Phacotaceae sensu stricto (Phacotus, Pteromonas, and Wislouchiella) and Dysmorphococcus globosus. This indicates at least three independent origins of loricae within the Volvocales. © 2010 Japanese Society of Phycology.

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A new multicompartmental reaction-diffusion modeling method links transient membrane attachment of E. coli MinE to E-ring formation

Arjunan, S. N. V. and Tomita, M.

Syst Synth Biol. (Systems and Synthetic Biology) 4 ( 1 ) 35-53 - 53 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 18725325

Many important cellular processes are regulated by reaction-diffusion (RD) of molecules that takes place both in the cytoplasm and on the membrane. To model and analyze such multicompartmental processes, we developed a lattice-based Monte Carlo method, Spatiocyte that supports RD in volume and surface compartments at single molecule resolution. Stochasticity in RD and the excluded volume effect brought by intracellular molecular crowding, both of which can significantly affect RD and thus, cellular processes, are also supported. We verified the method by comparing simulation results of diffusion, irreversible and reversible reactions with the predicted analytical and best available numerical solutions. Moreover, to directly compare the localization patterns of molecules in fluorescence microscopy images with simulation, we devised a visualization method that mimics the microphotography process by showing the trajectory of simulated molecules averaged according to the camera exposure time. In the rod-shaped bacterium Escherichia coli, the division site is suppressed at the cell poles by periodic pole-to-pole oscillations of the Min proteins (MinC, MinD and MinE) arising from carefully orchestrated RD in both cytoplasm and membrane compartments. Using Spatiocyte we could model and reproduce the in vivo MinDE localization dynamics by accounting for the previously reported properties of MinE. Our results suggest that the MinE ring, which is essential in preventing polar septation, is largely composed of MinE that is transiently attached to the membrane independently after recruited by MinD. Overall, Spatiocyte allows simulation and visualization of complex spatial and reaction-diffusion mediated cellular processes in volumes and surfaces. As we showed, it can potentially provide mechanistic insights otherwise difficult to obtain experimentally. © 2009 The Author(s).

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Computational prediction and experimental validation of evolutionarily conserved microRNA target genes in bilaterian animals

Takane, K., Fujishima, K., Watanabe, Y., Sato, A., Saito, N., Tomita, M and Kanai, A.

BMC Genomics (BMC Genomics) 11 ( 1 ) 101 2010

Research paper (scientific journal), Joint Work, Accepted

Background: In many eukaryotes, microRNAs (miRNAs) bind to complementary sites in the 3'-untranslated regions (3'-UTRs) of target messenger RNAs (mRNAs) and regulate their expression at the stage of translation. Recent studies have revealed that many miRNAs are evolutionarily conserved; however, the evolution of their target genes has yet to be systematically characterized. We sought to elucidate a set of conserved miRNA/target-gene pairs and to analyse the mechanism underlying miRNA-mediated gene regulation in the early stage of bilaterian evolution.Results: Initially, we extracted five evolutionarily conserved miRNAs (let-7, miR-1, miR-124, miR-125/lin-4, and miR-34) among five diverse bilaterian animals. Subsequently, we designed a procedure to predict evolutionarily conserved miRNA/target-gene pairs by introducing orthologous gene information. As a result, we extracted 31 orthologous miRNA/target-gene pairs that were conserved among at least four diverse bilaterian animals; the prediction set showed prominent enrichment of orthologous miRNA/target-gene pairs that were verified experimentally. Approximately 84% of the target genes were regulated by three miRNAs (let-7, miR-1, and miR-124) and their function was classified mainly into the following categories: development, muscle formation, cell adhesion, and gene regulation. We used a reporter gene assay to experimentally verify the downregulation of six candidate pairs (out of six tested pairs) in HeLa cells.Conclusions: The application of our new method enables the identification of 31 miRNA/target-gene pairs that were expected to have been regulated from the era of the common bilaterian ancestor. The downregulation of all six candidate pairs suggests that orthologous information contributed to the elucidation of the primordial set of genes that has been regulated by miRNAs; it was also an efficient tool for the elimination of false positives from the predicted candidates. In conclusion, our study identified potentially important miRNA-target pairs that were evolutionarily conserved throughout diverse bilaterian animals and that may provide new insights into early-stage miRNA functions. © 2010 Takane et al; licensee BioMed Central Ltd.

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A Comprehensive Resource of Interacting Protein Regions for Refining Human Transcription Factor Networks

Sato, M. E., Fujimori, S., Ishizaka, M., Hirai1, N., Masuoka1, K., Saito, R., Ozawa, Y., Hino, K., Washio, T., Tomita, M., Yamashita, T., Oshikubo, T., Akasaka, H., Sugiyama, J., Matsumoto, Y. and Yanagawa, H.

PLoS ONE (PLoS ONE) 5 ( 2 ) e9289 2010

Research paper (scientific journal), Joint Work, Accepted

Large-scale data sets of protein-protein interactions (PPIs) are a valuable resource for mapping and analysis of the topological and dynamic features of interactome networks. The currently available large-scale PPI data sets only contain information on interaction partners. The data presented in this study also include the sequences involved in the interactions (i.e., the interacting regions, IRs) suggested to correspond to functional and structural domains. Here we present the first large-scale IR data set obtained using mRNA display for 50 human transcription factors (TFs), including 12 transcription-related proteins. The core data set (966 IRs; 943 PPIs) displays a verification rate of 70%. Analysis of the IR data set revealed the existence of IRs that interact with multiple partners. Furthermore, these IRs were preferentially associated with intrinsic disorder. This finding supports the hypothesis that intrinsically disordered regions play a major role in the dynamics and diversity of TF networks through their ability to structurally adapt to and bind with multiple partners. Accordingly, this domain-based interaction resource represents an important step in refining protein interactions and networks at the domain level and in associating network analysis with biological structure and function. © 2010 Miyamoto-Sato et al.

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Chlorogonium complexum sp. nov. (Volvocales, Chlorophyceae), and morphological evolution of Chlorogonium

Nakada, T., Soga, T., Tomita, M. and Nozaki, H.

Eur. J. Phycol. 45 ( 1 ) 97-107 - 106 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 09670262

We isolated strains of a new species of Chlorogonium (Cg.), Cg. complexum Nakada sp. nov. The genus Chlorogonium was recently emended to have several, irregularly distributed contractile vacuoles, two-to-many pyrenoids associated with fragmented starch plates and tubular thylakoid lamellae penetrating their matrices, and stigma composed of a single layer of globules. Chlorogonium complexum has these diagnostic characters of the emended genus. It is distinguished from other species of Chlorogonium, Cg. euchlorum, Cg. elongatum and Cg. capillatum, on the combination of diagnostic characters, such as the number and stability of pyrenoids and the mode of encystment. Based on a dataset combining 18S rRNA, RuBisCO large subunit (rbcL) and P700 chl a-apoprotein A2 (psaB) gene sequences, the phylogenetic position of Chlorogonium was determined. Chlorogonium complexum represented an independent lineage, sister to Cg. euchlorum and Cg. capillatum. The evolution of diagnostic characters among Chlorogonium species is discussed based on the molecular phylogenetic analyses, and a key to species of Chlorogonium and fusiform chlamydomonads with irregularly distributed contractile vacuoles is presented. © 2010 British Phycological Society.

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Computational prediction of nucleosome positioning by calculating the relative fragment frequency index of nucleosomal sequences

Ogawa, R., Kitagawa, N., Ashida, H., Saito, R. and Tomita, M.

FEBS Letters (FEBS Letters) 584 ( 8 ) 1498-1502 - 1502 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 00145793

We developed an accurate method to predict nucleosome positioning from genome sequences by refining the previously developed method of Peckham et al. (2007) [19]. Here, we used the relative fragment frequency index we developed and a support vector machine to screen for nucleosomal and linker DNA sequences. Our twofold cross-validation revealed that the accuracy of our method based on the area under the receiver operating characteristic curve was 81%, whereas that of Peckham's method was 75% when both of two nucleosomal sequence data obtained from independent experiments were used for validation. We suggest that our method is more effective in predicting nucleosome positioning. © 2010 Federation of European Biochemical Societies.

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One-Dimensional Capillary Liquid Chromatographic Separation Coupled with Tandem Mass Spectrometry Unveils the Escherichia coli Proteome on a Microarray Scale

Iwasaki, M., Miwa, S., Ikegami, T., Tomita, M., Tanaka, N. and Ishihama, Y.

Anal. Chem. (Analytical Chemistry) 82 ( 7 ) 2616-2620 - 2620 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 00032700

We successfully identified the proteome expressed in Escherichia coli (E. coli) cells on a microarray scale using one-dimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a 350 cm long, 100 μm i. d., monolithic silica-C18 capillary column. E. coli tryptic digest (4 μg) was injected onto the column, and a 41 h gradient was applied with a flow rate of 500 nL/min at less than 20 MPa. In total, 22 196 nonredundant tryptic peptides from 2602 proteins, including 830 membrane proteins, were identified from the E. coli cells (triplicate analysis), in which an equivalent number of genes was detected by transcriptome analysis. Approximately a 5-fold larger peak response on average was obtained in this system, compared with that obtained by conventional capillary LC-MS/MS analysis with a 15 cm long, 3 μm diameter C18 silica particle-packed column. The higher response suggests that the influence of ionization suppression was drastically reduced by the high-efficiency separation on the long monolithic silica column coupled with the shallow gradient. Because this high-resolution system does not require any additional separation prior to LC-MS/MS, this "one-shot" proteomics approach can simplify the workflow of shotgun proteomics and minimize the sample amount, as well as reduce the total analysis time, despite the use of prolonged shallow gradient elution. © 2010 American Chemical Society.

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Gene knockout and metabolome analysis of carnitine/organic cation transporter OCTN1

Kato, Y., Kubo, Y., Iwata, D., Kato, S., Sudo, T., Sugiura, T., Kagaya, T., Wakayama, T.,Hirayama, A., Sugimoto, M., Sugihara, K., Kaneko, S., Soga, T., Asano, M., Tomita, M., Matsui, T., Wada, M. and Tsuji A.

Pharm Res 27 ( 5 ) 832-840 - 40 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 0724-8741

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Towards the systematic discovery of signal transduction networks using phosphorylation dynamics data

Imamura, H., Yachie, N., Saito, R., Ishihama, Y. and Tomita, M.

BMC Bioinformatics (BMC Bioinformatics) 11 232 2010

Research paper (scientific journal), Joint Work, Accepted

Background: Phosphorylation is a ubiquitous and fundamental regulatory mechanism that controls signal transduction in living cells. The number of identified phosphoproteins and their phosphosites is rapidly increasing as a result of recent mass spectrometry-based approaches.Results: We analyzed time-course phosphoproteome data obtained previously by liquid chromatography mass spectrometry with the stable isotope labeling using amino acids in cell culture (SILAC) method. This provides the relative phosphorylation activities of digested peptides at each of five time points after stimulating HeLa cells with epidermal growth factor (EGF). We initially calculated the correlations between the phosphorylation dynamics patterns of every pair of peptides and connected the strongly correlated pairs to construct a network. We found that peptides extracted from the same intracellular fraction (nucleus vs. cytoplasm) tended to be close together within this phosphorylation dynamics-based network. The network was then analyzed using graph theory and compared with five known signal-transduction pathways. The dynamics-based network was correlated with known signaling pathways in the NetPath and Phospho.ELM databases, and especially with the EGF receptor (EGFR) signaling pathway. Although the phosphorylation patterns of many proteins were drastically changed by the EGF stimulation, our results suggest that only EGFR signaling transduction was both strongly activated and precisely controlled.Conclusions: The construction of a phosphorylation dynamics-based network provides a useful overview of condition-specific intracellular signal transduction using quantitative time-course phosphoproteome data under specific experimental conditions. Detailed prediction of signal transduction based on phosphoproteome dynamics remains challenging. However, since the phosphorylation profiles of kinase-substrate pairs on the specific pathway were localized in the dynamics-based network, our method will be a complementary strategy to explore new components of protein signaling pathways in combination with previous methods (including software) of predicting direct kinase-substrate relationships. © 2010 Imamura et al; licensee BioMed Central Ltd.

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Transcription-Associated Mutagenesis Increases Protein Sequence Diversity More Effectively than Does Random Mutagenesis in Escherichia coli

Kim, H., Lee, B. S., Tomita, M. and Kanai, A.

PLoS ONE (PLoS ONE) 5 ( 5 ) e10567 2010

Research paper (scientific journal), Joint Work, Accepted

Background: During transcription, the nontranscribed DNA strand becomes single-stranded DNA (ssDNA), which can form secondary structures. Unpaired bases in the ssDNA are less protected from mutagens and hence experience more mutations than do paired bases. These mutations are called transcription-associated mutations. Transcription-associated mutagenesis is increased under stress and depends on the DNA sequence. Therefore, selection might significantly influence protein-coding sequences in terms of the transcription-associated mutability per transcription event under stress to improve the survival of Escherichia coli. Methodology/Principal Findings: The mutability index (MI) was developed by Wright et al. to estimate the relative transcription-associated mutability of bases per transcription event. Using the most stable fold of each ssDNA that have an average length n, MI was defined as (the number of folds in which the base is unpaired)/n ×(highest -ΔG of all n folds in which the base is unpaired), where ΔG is the free energy. The MI values show a significant correlation with mutation data under stress but not with spontaneous mutations in E. coli. Protein sequence diversity is preferred under stress but not under favorable conditions. Therefore, we evaluated the selection pressure on MI in terms of the protein sequence diversity for all the protein-coding sequences in E. coli. The distributions of the MI values were lower at bases that could be substituted with each of the other three bases without affecting the amino acid sequence than at bases that could not be so substituted. Start codons had lower distributions of MI values than did nonstart codons. Conclusions/Significance: Our results suggest that the majority of protein-coding sequences have evolved to promote protein sequence diversity and to reduce gene knockout under stress. Consequently, transcription-associated mutagenesis increases protein sequence diversity more effectively than does random mutagenesis under stress. Nonrandom transcription-associated mutagenesis under stress should improve the survival of E. coli. © 2010 Kim et al.

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Core promoter structure and genomic context reflect histone 3 lysine 9 acetylation patterns

Kratz, A., Arner, E., Saito, R., Kubosaki, A., Kawai, J., Suzuki, H., Carninci, P., Arakawa, T., Tomita, M., Hayashizaki, Y. and Daub, C.O.

BMC Genomics (BMC Genomics) 11 ( 1 ) 257 2010

Research paper (scientific journal), Joint Work, Accepted

Background: Histone modifications play an important role in gene regulation. Acetylation of histone 3 lysine 9 (H3K9ac) is generally associated with transcription initiation and unfolded chromatin, thereby positively influencing gene expression. Deep sequencing of the 5' ends of gene transcripts using DeepCAGE delivers detailed information about the architecture and expression level of gene promoters. The combination of H3K9ac ChIP-chip and DeepCAGE in a myeloid leukemia cell line (THP-1) allowed us to study the spatial distribution of H3K9ac around promoters using a novel clustering approach. The promoter classes were analyzed for association with relevant genomic sequence features.Results: We performed a clustering of 4,481 promoters according to their surrounding H3K9ac signal and analyzed the clustered promoters for association with different sequence features. The clustering revealed three groups with major H3K9ac signal upstream, centered and downstream of the promoter. Narrow single peak promoters tend to have a concentrated activity of H3K9ac in the upstream region, while broad promoters tend to have a concentrated activity of H3K9ac and RNA polymerase II binding in the centered and downstream regions. A subset of promoters with high gene expression level, compared to subsets with low and medium gene expression, shows dramatic increase in H3K9ac activity in the upstream cluster only; this may indicate that promoters in the centered and downstream clusters are predominantly regulated at post-initiation steps. Furthermore, the upstream cluster is depleted in CpG islands and more likely to regulate un-annotated genes.Conclusions: Clustering core promoters according to their surrounding acetylation signal is a promising approach for the study of histone modifications. When examining promoters clustered into groups according to their surrounding H3K9 acetylation signal, we find that the relative localization and intensity of H3K9ac is very specific depending on characteristic sequence features of the promoter. Experimental data from DeepCAGE and ChIP-chip experiments using undifferentiated (monocyte) and differentiated (macrophage) THP-1 cells leads us to the same conclusions. © 2010 Kratz et al; licensee BioMed Central Ltd.

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Unveiling cellular biochemical reactions via metabolomics-driven approaches

Saito, N., Ohashi, Y., Soga, T. and Tomita, M.

Curr Opin Microbiol. (Current Opinion in Microbiology) 13 ( 3 ) 358-362 - 362 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 13695274

Despite the widespread availability of genome sequences and the systematic assignment of individual open reading frames, many of the activities of metabolic enzymes are still veiled. It is difficult to assign biochemical functions to enzymes and uncover the in vivo regulation of their enzymatic activities, but this is crucial to understand cellular functions. The development of metabolomics and metabolite profiling, which can globally analyze intracellular and extracellular metabolites, has provided some valuable tools for systems biology. This report reviews the recent metabolomics approaches to elucidate unidentified metabolic systems while focusing on their enzymatic activities, including their biochemical and biological functions, and their regulation in response to environmental perturbations in microorganisms. © 2010 Elsevier Ltd. All rights reserved.

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Complete genome sequence and comparative analysis of Shewanella violacea, a psychrophilic and piezophilic bacterium from deep sea floor sediments

Aono, E., Baba, T., Ara, T., Nishi, T., Nakamichi, T., Inamoto, E., Toyonaga, H., Hasegawa, M., Takai, Y., Okumura,Y., Baba, M., Tomita, M., Kato, C., Oshima, T., Nakasone, K. and Mori

Mol. BioSyst. (Molecular BioSystems) 6 ( 7 ) 1216-1226 - 1226 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 1742206X

Remineralization of organic matter in deep-sea sediments is important in oceanic biogeochemical cycles, and bacteria play a major role in this process. Shewanella violacea DSS12 is a psychrophilic and piezophilic γ-proteobacterium that was isolated from the surface layer of deep sea sediment at a depth of 5110 m. Here, we report the complete genome sequence of S. violacea and comparative analysis with the genome of S. oneidensis MR-1, isolated from sediments of a freshwater lake. Unlike S. oneidensis, this deep-sea Shewanella possesses very few terminal reductases for anaerobic respiration and no c-type cytochromes or outer membrane proteins involved in respiratory Fe(iii) reduction, which is characteristic of most Shewanella species. Instead, the S. violacea genome contains more terminal oxidases for aerobic respiration and a much greater number of putative secreted proteases and polysaccharases, in particular, for hydrolysis of collagen, cellulose and chitin, than are encoded in S. oneidensis. Transporters and assimilatory reductases for nitrate and nitrite, and nitric oxide-detoxifying mechanisms (flavohemoglobin and flavorubredoxin) are found in S. violacea. Comparative analysis of the S. violacea genome revealed the respiratory adaptation of this bacterium to aerobiosis, leading to predominantly aerobic oxidation of organic matter in surface sediments, as well as its ability to efficiently use diverse organic matter and to assimilate inorganic nitrogen as a survival strategy in the nutrient-poor deep-sea floor. © The Royal Society of Chemistry 2010.

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Differential metabolomics software for capillary electrophoresis-mass spectrometry data analysis

Sugimoto, Masahiro ; Hirayama, Akiyoshi ; Ishikawa, Takamasa ; Robert, Martin ; Baran, Richard ; Uehara, Keizo ; Kawai, Katsuya ; Soga, Tomoyoshi ; Tomita, Masaru

METABOLOMICS 6 ( 1 ) 27-41 - 41 2010

Research paper (scientific journal), Joint Work, ISSN 1573-3882

In metabolomics, the rapid identification of quantitative differences between multiple biological samples remains a major challenge. While capillary electrophoresis-mass spectrometry (CE-MS) is a powerful tool to simultaneously quantify charged metabolites, reliable and easy-to-use software that is well suited to analyze CE-MS metabolic profiles is still lacking. Optimized software tools for CE-MS are needed because of the sometimes large variation in migration time between runs and the wider variety of peak shapes in CE-MS data compared with LC-MS or GC-MS. Therefore, we implemented a stand-alone application named JDAMP (Java application for Differential Analysis of Metabolite Profiles), which allows users to identify the metabolites that vary between two groups. The main features include fast calculation modules and a file converter using an original compact file format, baseline subtraction, dataset normalization and alignment, visualization on 2D plots (m/z and time axis) with matching metabolite standards, and the detection of significant differences between metabolite profiles. Moreover, it features an easy-to-use graphical user interface that requires only a few mouse-actions to complete the analysis. The interface also enables the analyst to evaluate the semiautomatic processes and interactively tune options and parameters depending on the input datasets. The confirmation of findings is available as a list of overlaid electropherograms, which is ranked using a novel difference-evaluation function that accounts for peak size and distortion as well as statistical criteria for accurate difference-detection. Overall, the JDAMP software complements other metabolomics data processing tools and permits easy and rapid detection of significant differences between multiple complex CE-MS profiles. © Springer Science+Business Media, LLC 2009.

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Another Origin of Coloniality in Volvocaleans: The Phylogenetic Position of Pyrobotrys Arnoldi (Spondylomoraceae, Volvocales)

Nakada, T., Nozaki, H. and Tomita, M.

J. Eukaryot. Microbiol. (Journal of Eukaryotic Microbiology) 57 ( 4 ) 379-382 - 382 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 10665234

Colonial volvocaleans (Chlorophyceae) are used as a standard model of multicellular evolution. However, the phylogenetic position of the colonial volvocalean family Spondylomoraceae has yet to be resolved. To examine this, the molecular phylogenies of Pyrobotrys stellata and Pyrobotrys squarrosa were analyzed using combined 18S rRNA, RUBISCO large subunit, and P700 chl a-apoprotein A2 gene sequences. In the phylogenetic trees, Pyrobotrys belonged to the clade Caudivolvoxa and was not closely related to other colonial volvocalean flagellates. The results indicate that colony formation of Spondylomoraceae independently evolved from unicellular volvocaleans. The phylogenetic position of problematic "Pascherina tetras" SAG 159-1 was also analyzed. © 2010 by the International Society of Protistologists.

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Large-scale comparative phosphoproteomics identifies conserved phosphorylation sites in plants

Nakagami, H., Sugiyama, N., Mochida, K., Daudi, A., Yoshida, Y., Toyoda, T., Tomita, M., Ishihama, Y. and Shirasu, K.

Plant Physiology (Plant Physiology) 153 ( 3 ) 1161-1174 - 1174 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 00320889

Knowledge of phosphorylation events and their regulation is crucial to understand the functional biology of plants. Here, we report a large-scale phosphoproteome analysis in the model monocot rice (Oryza sativa japonica 'Nipponbare'), an economically important crop. Using unfractionated whole-cell lysates of rice cells, we identified 6,919 phosphopeptides from 3,393 proteins. To investigate the conservation of phosphoproteomes between plant species, we developed a novel phosphorylation-site evaluation method and performed a comparative analysis of rice and Arabidopsis (Arabidopsis thaliana). The ratio of tyrosine phosphorylation in the phosphoresidues of rice was equivalent to those in Arabidopsis and human. Furthermore, despite the phylogenetic distance and the use of different cell types, more than 50% of the phosphoproteins identified in rice and Arabidopsis, which possessed ortholog(s), had an orthologous phosphoprotein in the other species. Moreover, nearly half of the phosphorylated orthologous pairs were phosphorylated at equivalent sites. Further comparative analyses against the Medicago phosphoproteome also showed similar results. These data provide direct evidence for conserved regulatory mechanisms based on phosphorylation in plants. We also assessed the phosphorylation sites on nucleotide-binding leucine-rich repeat proteins and identified novel conserved phosphorylation sites that may regulate this class of proteins. © 2010 American Society of Plant Biologists.

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Prediction of metabolite identity from accurate mass, migration time prediction and isotopic pattern information in CE-TOFMS data

Sugimoto, M., Hirayama, A., Robert, M., Abe, S., Soga, T. and Tomita, M.

Electrophoresis (Electrophoresis) 31 ( 14 ) 2311–2318 - 2318 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 01730835

CE-TOFMS is a powerful method for profiling charged metabolites. However, the limited availability of metabolite standards hinders the process of identifying compounds from detected features in CE-TOFMS data sets. To overcome this problem, we developed a method to identify unknown peaks based on the predicted migration time (tm) and accurate m/z values. We developed a predictive model using 375 standard cationic metabolites and support vector regression. The model yielded good correlations between the predicted and measured tm (R=0.952 and 0.905 using complete and cross-validation data sets, respectively). Using the trained model, we subsequently predicted the tm for 2938 metabolites available from the public databases and assigned tentative identities to noise-filtered features in human urine samples. While 38.9% of the peaks were assigned metabolite names by matching with the standard library alone, the proportion increased to 52.2%. The proposed methodology increases the value of metabolomic data sets obtained from CE-TOFMS profiling. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

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Protein complex prediction via verifying and reconstructing the topology of domain-domain interactions

Ozawa, Y., Saito, R., Fujimori, S., Kashima, H., Ishizaka, Hiroshi Yanagawa, H., Miyamoto-Sato, E. and Tomita, M.

BMC Bioinformatics (BMC Bioinformatics) 11 350 2010

Research paper (scientific journal), Joint Work, Accepted

Background: High-throughput methods for detecting protein-protein interactions enable us to obtain large interaction networks, and also allow us to computationally identify the associations of proteins as protein complexes. Although there are methods to extract protein complexes as sets of proteins from interaction networks, the extracted complexes may include false positives because they do not account for the structural limitations of the proteins and thus do not check that the proteins in the extracted complex can simultaneously bind to each other. In addition, there have been few searches for deeper insights into the protein complexes, such as of the topology of the protein-protein interactions or into the domain-domain interactions that mediate the protein interactions.Results: Here, we introduce a combinatorial approach for prediction of protein complexes focusing not only on determining member proteins in complexes but also on the DDI/PPI organization of the complexes. Our method analyzes complex candidates predicted by the existing methods. It searches for optimal combinations of domain-domain interactions in the candidates based on an assumption that the proteins in a candidate can form a true protein complex if each of the domains is used by a single protein interaction. This optimization problem was mathematically formulated and solved using binary integer linear programming. By using publicly available sets of yeast protein-protein interactions and domain-domain interactions, we succeeded in extracting protein complex candidates with an accuracy that is twice the average accuracy of the existing methods, MCL, MCODE, or clustering coefficient. Although the configuring parameters for each algorithm resulted in slightly improved precisions, our method always showed better precision for most values of the parameters.Conclusions: Our combinatorial approach can provide better accuracy for prediction of protein complexes and also enables to identify both direct PPIs and DDIs that mediate them in complexes. © 2010 Ozawa et al; licensee BioMed Central Ltd.

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Chemical dephosphorylation for identification of multiply phosphorylated peptides and phosphorylation site determination

Kyono, Y., Sugiyama, N., Tomita, M. and Ishihama, Y.

Rapid Commun. Mass Spectrom. (Rapid Communications in Mass Spectrometry) 24 ( 15 ) 2277–2282 - 2282 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 09514198

We have developed a novel strategy to improve the efficiency of identification of multiply phosphorylated peptides isolated by hydroxy acid modified metal oxide chromatography (HAMMOC). This strategy consists of alkali-induced chemical dephosphorylation (beta-elimination reaction) of phosphopeptides isolated by HAMMOC prior to analysis by liquid chromatography/mass spectrometry (LC/MS). This approach identified 1.9-fold more multiply phosphorylated peptides than the conventional approach without beta-elimination from a digested mixture of three standard phosphoproteins. In addition, the accuracy of phosphorylation site determination in synthetic phosphopeptides was significantly improved. Finally, we applied this approach to a cell lysate. By combining this dephosphorylation approach with the conventional approach, we successfully identified 1649 unique phosphopeptides, including 325 multiply phosphorylated phosphopeptides, from 200mg of cultured Arabidopsis cells. These results indicate that chemical dephosphorylation prior to LC/MS analysis increases the efficiency of identification of multiply phosphorylated peptides, as well as the accuracy of phosphorylation site determination. © 2010 John Wiley & Sons, Ltd.

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Metabolomic Profiles and Sensory Attributes of Edamame under Various Storage Duration and Temperature Conditions.

Sugimoto, M., Goto, H., Otomo, K., Ito, M., Onuma, H., Suzuki, A., Sugawara, M., Abe, S., Tomita, M. and Soga, T.

J Agric Food Chem. (Journal of Agricultural and Food Chemistry) 58 ( 14 ) 8418–8425 - 8425 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 00218561

Its high nutritional content and sensory characteristics make edamame a popular vegetable bean. However, because of its short shelf-life, it is important to optimize the storage conditions to maintain its quality during distribution to consumers. We focused on storage conditions to investigate the temporal changes in the metabolic profiles and sensory characteristics of edamame during transportation from the site of harvest to the site of purchase/consumption. We conducted metabolomic analysis and sensory evaluation tests of edamame stored for different lengths and at different temperatures. Charged metabolites were profiled by capillary electrophoresis-mass spectrometry, and free sugars were quantified by liquid chromatography-tandem mass spectrometry. In comparison to the gradual decrease in its sensory characteristics over time, the changes in metabolite profiles manifested four different patterns. In particular, changes in amino acid levels were related to sensory attributes. The downstream metabolites of shikimate as well as phospholipids and γ-aminobutyric acid increased with increasing storage temperatures. © 2010 American Chemical Society.

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